Quantifying the probability of a successful marine bioinvasion due to source-destination risk factors.

The increasing spread of marine non-indigenous species (NIS) due to the growth in global shipping traffic is causing widespread concern for the ecological and economic impacts of marine bioinvasions. Risk management authorities need tools to identify pathways and source regions of priority concern to better target efforts for preventing NIS introduction. The probability of a successful NIS introduction is affected by the likelihood that a marine species entrained in a transport vector will survive the voyage between origin and destination locations and establish an independently reproducing population at the destination. Three important risk factors are voyage duration, range of environmental conditions encountered during transit and environmental similarity between origin and destination. In this study, we aimed for a globally comprehensive approach to assembling quantifications of source-destination risk factors from every potential origin to every potential destination. To derive estimates of voyage-related marine biosecurity risk, we used computer-simulated vessel paths between pairs of ecoprovinces in the Marine Ecoregions Of the World biogeographic classification system. We used the physical length of each path to calculate voyage duration risk and the cross-latitudinal extent of the path to calculate voyage path risk. Environmental similarity risk was based on comparing annual average sea surface temperature and salinity within each ecoprovince to those of other ecoprovinces. We derived three separate sets of risk quantifications, one each for voyage duration, voyage path and environmental similarity. Our quantifications can be applied to studies that require source-destination risk estimates. They can be used separately or combined, depending on the importance of the types of source-destination risks that might be relevant to particular scientific or risk management questions or applications.

Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR.

Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non-indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time-demanding, and thereby can hinder management efforts and restrict the "opportunity window" for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species-specific free-floating extra-cellular eDNA (free-eDNA) signals, i.e., detection of species-specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof-of-concept aquarium study was conducted with three distinct marine species: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analysing aquarium marine water samples using an optimized species-specific ddPCR assay. The results demonstrated the consistent detection of S. spallanzanii and B. neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24-72 h. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point-of-need detection and rapid management responses.

An investigation into the stability and degradation of plastics in aquatic environments using a large-scale field-deployment study.

The fragmentation of plastic debris is a key pathway to the formation of microplastic pollution. These disintegration processes depend on the materials' physical and chemical characteristics, but insight into these interrelationships is still limited, especially under natural conditions. Five plastics of known polymer/additive compositions and processing histories were deployed in aquatic environments and recovered after six and twelve months. The polymer types used were linear low density polyethylene (LLDPE), oxo-degradable LLDPE (oxoLLDPE), poly(ethylene terephthalate) (PET), polyamide-6 (PA6), and poly(lactic acid) (PLA). Four geographically distinct locations across Aotearoa/New Zealand were chosen: three marine sites and a wastewater treatment plant (WWTP). Accelerated UV-weathering under controlled laboratory conditions was also carried out to evaluate artificial ageing as a model for plastic degradation in the natural environment. The samples' physical characteristics and surface microstructures were studied for each deployment location and exposure time. The strongest effects were found for oxoLLDPE upon artificial ageing, with increased crystallinity, intense surface cracking, and substantial deterioration of its mechanical properties. However, no changes to the same extent were found after recovery of the deployed material. In the deployment environments, the chemical nature of the plastics was the most relevant factor determining their behaviours. Few significant differences between the four aquatic locations were identified, except for PA6, where indications for biological surface degradation were found only in seawater, not the WWTP. In some cases, artificial ageing reasonably mimicked the changes which some plastic properties underwent in aquatic environments, but generally, it was no reliable model for natural degradation processes. The findings from this study have implications for the understanding of the initial phases of plastic degradation in aquatic environments, eventually leading to microplastics formation. They can also guide the interpretation of accelerated laboratory ageing for the fate of aquatic plastic pollution, and for the testing of aged plastic samples.

Harnessing environmental DNA to reveal biogeographical patterns of non-indigenous species for improved co-governance of the marine environment in Aotearoa New Zealand.

Aotearoa New Zealand's Northern region is a major gateway for the incursion and establishment of non-indigenous species (NIS) populations due to high numbers of recreational and commercial vessels. This region also holds a unique marine ecosystem, home to many taonga (treasured) species of cultural and economic importance. Regular surveillance, eradication plans and public information sharing are undertaken by local communities and governmental organizations to protect these ecosystems from the impact of NIS. Recently, considerable investments went into environmental DNA (eDNA) research, a promising approach for the early detection of NIS for complementing existing biosecurity systems. We applied eDNA metabarcoding for elucidating bioregional patterns of NIS distributions across a gradient from harbors (NIS hotspots) to open seas (spreading areas). Samples were collected during a research cruise sailing across three Aotearoa New Zealand harbors, Waitemata, Whangarei and Pewhairangi (Bay of Islands), and their adjacent coastal waters. The small-ribosomal subunit (18S rRNA) and mitochondrial cytochrome c oxidase I (COI) genes were screened using the online Pest Alert Tool for automated detection of putative NIS sequences. Using a probabilistic modelling approach, location-dependent occupancies of NIS were investigated and related to the current information on species distribution from biosecurity surveillance programs. This study was collaboratively designed with Maori partners to initiate a model of co-governance within the existing science system.

Hurdles and opportunities in implementing marine biosecurity systems in data-poor regions.

Managing marine nonindigenous species (mNIS) is challenging, because marine environments are highly connected, allowing the dispersal of species across large spatial scales, including geopolitical borders. Cross-border inconsistencies in biosecurity management can promote the spread of mNIS across geopolitical borders, and incursions often go unnoticed or unreported. Collaborative surveillance programs can enhance the early detection of mNIS, when response may still be possible, and can foster capacity building around a common threat. Regional or international databases curated for mNIS can inform local monitoring programs and can foster real-time information exchange on mNIS of concern. When combined, local species reference libraries, publicly available mNIS databases, and predictive modeling can facilitate the development of biosecurity programs in regions lacking baseline data. Biosecurity programs should be practical, feasible, cost-effective, mainly focused on prevention and early detection, and be built on the collaboration and coordination of government, nongovernment organizations, stakeholders, and local citizens for a rapid response.

CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning.

Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest advancements involves utilizing CRISPR-Cas technology to build programmable, rapid, ultrasensitive, and specific diagnostics. CRISPR-based diagnostics (CRISPR-Dx) has the potential to allow robust, reliable, and cost-effective biomonitoring in near real time. However, several challenges must be overcome before CRISPR-Dx can be established as a mainstream tool for marine biomonitoring. A critical unmet challenge is the need to design, optimize, and experimentally validate CRISPR-Dx assays. Artificial intelligence has recently been presented as a potential approach to tackle this challenge. This perspective synthesizes recent advances in CRISPR-Dx and machine learning modeling approaches, showcasing CRISPR-Dx potential to progress as a rising molecular tool candidate for marine biomonitoring applications.

A spatio-temporal analysis of marine diatom communities associated with pristine and aged plastics.

Complex microbial communities colonize plastic substrates over time, strongly influencing their fate and potential impacts on marine ecosystems. Among the first colonizers, diatoms play an important role in the development of this 'plastiphere'. We investigated 936 biofouling samples and the factors influencing diatom communities associated with plastic colonization. These factors included geographic location (up to 800 km apart), duration of substrate submersion (1 to 52 weeks), plastics (5 polymer types) and impact of artificial ageing with UV light. Diatom communities colonizing plastic debris were primarily determined by their geographic location and submersion time, with the strongest changes occurring within two weeks of submersion. Several taxa were identified as early colonizers (e.g. Cylindrotheca, Navicula and Nitzschia spp.) with known strong adhesion capabilities. To a lesser extent, plastic-type and UV-ageing significantly affected community composition, with 14 taxa showing substrate-specificity. This study highlights the role of plastics types-state for colonization in the ocean.

Optimal sample type and number vary in small shallow lakes when targeting non-native fish environmental DNA.

Non-native fish have been shown to have deleterious impacts on freshwater ecosystems in New Zealand. Early detection is critical for their effective management. Traditional capture-based techniques may not detect newly introduced fish, especially if they are present in low abundance. Molecular techniques that target environmental DNA (eDNA) have been shown, in many instances, to be more sensitive, cost-effective and require lower sampling effort. However, appropriate sampling strategies are needed to ensure robust and interpretable data are obtained. In this study we used droplet digital PCR assays to investigate the presence of two non-native fish in New Zealand, the European perch (Perca fluviatilis) and rudd (Scardinius erythrophthalmus) in three small lakes. Samples were collected from water and surface sediment at near-shore and mid-lake sites. Probabilistic modelling was used to assess the occupancy of fish eDNA and develop guidance on sampling strategies. Based on the detection probability measures from the present study, at least six sites and five replicates per site are needed to reliably detect fish eDNA in sediment samples, and twelve sites with eight replicates per site for water samples. The results highlight the potential of developing monitoring and surveillance programs adapted to lakes, that include the use of assays targeting eDNA. This study focused on small shallow lakes, and it is likely that these recommendations may vary in larger, deeper, and more geomorphologically complex lakes, and this requires further research.

Pest Alert Tool-a web-based application for flagging species of concern in metabarcoding datasets.

Advances in high-throughput sequencing (HTS) technologies and their increasing affordability have fueled environmental DNA (eDNA) metabarcoding data generation from freshwater, marine and terrestrial ecosystems. Research institutions worldwide progressively employ HTS for biodiversity assessments, new species discovery and ecological trend monitoring. Moreover, even non-scientists can now collect an eDNA sample, send it to a specialized laboratory for analysis and receive in-depth biodiversity record from a sampling site. This offers unprecedented opportunities for biodiversity assessments across wide temporal and spatial scales. The large volume of data produced by metabarcoding also enables incidental detection of species of concern, including non-indigenous and pathogenic organisms. We introduce an online app-Pest Alert Tool-for screening nuclear small subunit 18S ribosomal RNA and mitochondrial cytochrome oxidase subunit I datasets for marine non-indigenous species as well as unwanted and notifiable marine organisms in New Zealand. The output can be filtered by minimum length of the query sequence and identity match. For putative matches, a phylogenetic tree can be generated through the National Center for Biotechnology Information's BLAST Tree View tool, allowing for additional verification of the species of concern detection. The Pest Alert Tool is publicly available at https://pest-alert-tool-prod.azurewebsites.net/.

Net overboard: Comparing marine eDNA sampling methodologies at sea to unravel marine biodiversity.

Environmental DNA (eDNA) analyses are powerful for describing marine biodiversity but must be optimized for their effective use in routine monitoring. To maximize eDNA detection probabilities of sparsely distributed populations, water samples are usually concentrated from larger volumes and filtered using fine-pore membranes, often a significant cost-time bottleneck in the workflow. This study aimed to streamline eDNA sampling by investigating plankton net versus bucket sampling, direct versus sequential filtration including self-preserving filters. Biodiversity was assessed using metabarcoding of the small ribosomal subunit (18S rRNA) and mitochondrial cytochrome c oxidase I (COI) genes. Multispecies detection probabilities were estimated for each workflow using a probabilistic occupancy modelling approach. Significant workflow-related differences in biodiversity metrics were reported. Highest amplicon sequence variant (ASV) richness was attained by the bucket sampling combined with self-preserving filters, comprising a large portion of microplankton. Less diversity but more metazoan taxa were captured in the net samples combined with 5 µm pore size filters. Prefiltered 1.2 µm samples yielded few or no unique ASVs. The highest average (~32%) metazoan detection probabilities in the 5 µm pore size net samples confirmed the effectiveness of preconcentration plankton for biodiversity screening. These results contribute to streamlining eDNA sampling protocols for uptake and implementation in marine biodiversity research and surveillance.

Quantifying impacts of human pressures on ecosystem services: Effects of widespread non-indigenous species in the Baltic Sea.

Ecosystem services (ES) are the benefits natural ecosystems provide to society, such as food provisioning, water supply, climate regulation and recreational benefits. Biological invasions are a major driver of global change, and several non-indigenous species (NIS) may alter key ecological feedbacks with ultimate consequences to ES, livelihoods and human wellbeing. Nonetheless, the effects of NIS on ES supply remain largely unquantified. Here we present the first quantitative case study assessing the impacts of widespread NIS on ES in the Baltic Sea, by developing and employing a robust and repeatable data-driven approach. All NIS with a sufficient knowledge base pose large and highly significant effects on ES, resulting on average 55 % change in the intensity of ES. Most impacts affected regulation services, concerning both abiotic and biotic realms, with little evidence on cultural and provisioning services. The methodology can be easily employed beyond the current study realm e.g. to better understand the roles of human pressures on ES in any ecosystem. Importantly, the study also identified major biases not only in the availability of taxonomic and sub-regional evidence, but also in the different study types employed to create the evidence base.

Application of multi-region input-output analysis to examine biosecurity risks associated with the global shipping network.

The vast majority of globally traded cargo is transported via maritime shipping. Whilst in port for loading and unloading, these ships can pick up local marine organisms with internal ballast water or as external biofouling assemblages and subsequently move these to destination far beyond their natural ranges. Over the past decades, this mechanism has led to the establishment of hundreds of non-indigenous species (NIS) around global coastlines. Marine NIS cause significant environmental, economic, cultural and human health impacts. Taking effective steps to preventing their dispersal and establishment is an enduring challenge for governments and conservation agencies around the world. Here we use international commodity trade data and a Nobel-Prize-winning economic analysis technique to develop a novel approach for assessing global marine NIS transfer risks. We show that by tracing the origins and destinations of seaborne trade connections, and the nature of the traded commodities, we can predict the strength of shipping vectors and associated marine biosecurity risks. We demonstrate the utility of our approach via a case-study, where we trace the spread of a hypothetical marine NIS from Japan and show the congruence of our model results with documented invasion histories from that region. Our study demonstrates that biosecurity risk can be assessed using established economic modelling frameworks on the basis of monetary transaction data alone, and without the need for detailed itineraries of the many thousand vessels making up the global commercial fleet. Novel, cost-effective tools are needed to mitigate biosecurity risks associated with maritime trade, and to meet conservation goals while enabling economic prosperity. The modelling framework presented here can be expanded to incorporate future risk factors, life-history traits of particular NIS of concern, and even adapted to simulate the dispersal of terrestrial pests or disease agents.

Does plastic type matter? Insights into non-indigenous marine larvae recruitment under controlled conditions.

Marine plastic debris (MPD) are a global threat to marine ecosystems. Among countless ecosystem impacts, MPD can serve as a vector for marine 'hitchhikers' by facilitating transport and subsequent spread of unwanted pests and pathogens. The transport and spread of these non-indigenous species (NIS) can have substantial impacts on native biodiversity, ecosystem services/functions and hence, important economic consequences. Over the past decade, increasing research interest has been directed towards the characterization of biological communities colonizing plastic debris, the so called Plastisphere. Despite remarkable advances in this field, little is known regarding the recruitment patterns of NIS larvae and propagules on MPD, and the factors influencing these patterns. To address this knowledge gap, we used custom-made bioassay chambers and ran four consecutive bioassays to compare the settlement patterns of four distinct model biofouling organisms' larvae, including the three notorious invaders Crassostrea gigas, Ciona savignyi and Mytilus galloprovincialis, along with one sessile macro-invertebrate Spirobranchus cariniferus, on three different types of polymers, namely Low-Linear Density Polyethylene (LLDPE), Polylactic Acid (PLA), Nylon-6, and a glass control. Control bioassay chambers were included to investigate the microbial community composition colonizing the different substrates using 16S rRNA metabarcoding. We observed species-specific settlement patterns, with larvae aggregating on different locations on the substrates. Furthermore, our results revealed that C. savignyi and S. cariniferus generally favoured Nylon and PLA, whereas no specific preferences were observed for C. gigas and M. galloprovincialis. We did not detect significant differences in bacterial community composition between the tested substrates. Taken together, our results highlight the complexity of interactions between NIS larvae and plastic polymers. We conclude that several factors and their potential interactions influenced the results of this investigation, including: (i) species-specific larval biological traits and ecology; (ii) physical and chemical composition of the substrates; and (iii) biological cues emitted by bacterial biofilm and the level of chemosensitivity of the different NIS larvae. To mitigate the biosecurity risks associated with drifting plastic debris, additional research effort is critical to effectively decipher the mechanisms involved in the recruitment of NIS on MPD.

Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.

Lake microbial communities are not resistant or resilient to repeated large-scale natural pulse disturbances.

Opportunities to study community-level responses to extreme natural pulse disturbances in unaltered ecosystems are rare. Lake sediment records that span thousands of years can contain well-resolved sediment pulses, triggered by earthquakes. These palaeorecords provide a means to study repeated pulse disturbances and processes of resistance (insensitivity to disturbance) and ecological resilience (capacity to regain structure, function and process). In this study, sedimentary DNA was extracted from a sediment core from Lake Paringa (New Zealand) that is situated in a near natural catchment. Metabarcoding and inferred functions were used to assess the lake microbial community over the past 1100 years - a period that included four major earthquakes. Microbial community composition and function differed significantly between highly perturbed (postseismic, ~50 years) phases directly after the earthquakes and more stable (interseismic, ~250 years) phases, indicating a lack of community resistance. Although community structure differed significantly in successive postseismic phases, function did not, suggesting potential functional redundancy. Significant differences in composition and function in successive interseismic phases demonstrate that communities are not resilient to large-scale natural pulse disturbances. The clear difference in structure and function, and high number of indicator taxa (responsible for driving differences in communities between phases) in the fourth interseismic phase probably represents a regime shift, possibly due to the two-fold increase in sediment and terrestrial biospheric organic carbon fluxes recorded following the fourth earthquake. Large pulse disturbances that enhance sediment inputs into lake systems may produce an underappreciated mechanism that destabilises lake ecosystem processes.

Meta-analysis on the ecological impacts of widely spread non-indigenous species in the Baltic Sea.

The introduction of non-indigenous species (NIS) is a major driver for global change in species biogeography, often associated with significant consequences for recipient ecosystems and services they provide for humans. Despite mandated by several high-level international legislative instruments, comprehensive quantitative evaluation on ecosystem impacts of marine NIS is scarce and lack a robust and data-driven assessment framework. The current study is aiming at fulfilling this gap, through quantitative assessment on the effects of the widespread NIS of the Baltic Sea on multiple ecosystem features and components including direct food-web effects. The outcomes of this study allowed identifying the most impacting widespread NIS, together with defining the processes underlying the most significant changes and outlined major sources of uncertainty. Lack and/or bias in the availability of evidence of impacts was recorded for several (both recent and early) introductions. Realizing a sophisticated, data and information-hungry framework for the evaluation of ecosystem impacts of NIS is not pragmatic for management purposes in the foreseeable future. Instead, simple approaches, such as application of common statistical parameters like absolute effect size, are more likely to result in tangible outcomes. As bearing no unit, effect sizes can be later easily aggregated across taxa, affected ecosystem features or spatial scales. The proposed approach enables performing systematic comparisons on the severity of impacts of different NIS along different study disciplines and ecosystems.

Poorer diversity but tougher species in old ballast water: Biosecurity challenges explored from visual and molecular techniques.

Millions of tons of water cross the oceans inside ships' ballast tanks every day. Planktonic species hitch-hike with water and some may pose risks to ecosystems and economies if get released and establish outside their native range. We monitored ballast water in different trans-equatorial travels, visually and using molecular techniques, and found significant increases of potential nuisance taxa over travel duration, despite evident diversity depletion. Thus, less diverse but more resistant and potentially more harmful communities persist in ballast water over long voyages. If we consider the enormous volume transported every day, the persistence of resistant species in ballast water would be threating the global marine biodiversity. This should be taken into account when modeling and assessing the bioinvasion risks associated with the ballast water and transfer considered in the future research.

Depicting Temporal, Functional, and Phylogenetic Patterns in Estuarine Diazotrophic Communities from Environmental DNA and RNA.

Seasonally nitrogen-limited and phosphorus-replete temperate coastal waters generally host dense and diverse diazotrophic communities. Despite numerous studies in marine systems, little is known about diazotrophs and their functioning in oligohaline estuarine environments. Here we applied a combination of nifH transcript and metagenomic shotgun sequencing approaches to investigate temporal shifts in taxonomic composition and nifH activity of size-fractionated diazotrophic communities in a shallow and mostly freshwater coastal lagoon. Patterns in active nifH phylotypes exhibited a clear seasonal succession, which reflected their different tolerances to temperature change and nitrogen (N) availability. Thus, in spring, heterotrophic diazotrophs (Proteobacteria) dominated the nifH phylotypes, while increasing water temperature and depletion of inorganic N fostered heterocystous Cyanobacteria in summer. Metagenomic data demonstrated four main N-cycling pathways and three of them with a clear seasonal pattern: denitrification (spring) → N2 fixation (summer) → assimilative NO3- reduction (fall), with NH4+ uptake into cells occurring across all seasons. Although a substantial denitrification signal was observed in spring, it could have originated from the re-suspended benthic rather than planktonic community. Our results contribute to a better understanding of the realized genetic potential of pelagic N2 fixation and its seasonal dynamics in oligohaline estuarine ecosystems, which are natural coastal biogeochemical reactors.

Stress resistance for unraveling potential biopollutants. Insights from ballast water community analysis through DNA.

In marine settings, anthropogenic disturbances and climate change increase the rate of biological invasions. Predicting still undescribed invasive alien species (IAS) is needed for preparing timely management responses. We tested a strategy for discovering new potential IAS using DNA in a trans-equatorial expedition onboard RV Polarstern. During one-month travel, species inside ballast water experienced oxygen depletion, warming, darkness and ammonium stress. Many organisms died but several phytoplankton and zooplankton survivors resisted and were detected through a robust combination of individual sampling, DNA barcoding and metabarcoding, new in ballast water studies. Ammonium was identified as an important influential factor to explain diversity changes in phytoplankton and zooplankton. Some species reproduced until the end of the travel. These species tolerant to travel stress could be targeted as potential IAS and prioritized for designing control measures. Introducing resistance to travel stress in biosecurity risk analysis would be recommended.

Biosecurity implications of drifting marine plastic debris: Current knowledge and future research.

The introduction and spread of marine non-indigenous species (NIS) and pathogens into new habitats are a major threat to biodiversity, ecosystem services, human health, and can have substantial economic consequences. Shipping is considered the main vector for marine biological invasions; less well understood is the increased spread of marine NIS and pathogens rafting on marine plastic debris (MPD). Despite an increasing research interest and recent progress in characterizing the plastisphere, this manuscript highlights critical knowledge gaps and research priorities towards a better understanding of the biosecurity implications of MPD. We advocate for future research to (i) investigate plastisphere community succession and the factors influencing NIS propagules and pathogens recruitment through robust experimental investigations; (ii) combine microscopy and molecular approaches to effectively assess the presence of specific taxa; (iii) include additional genetic markers to thoroughly characterize the biodiversity associated with MPD and explore the presence of specific marine pests.